Intra-articular gel for tissue and cartilage regeneration

Abstract

The present invention provides an intra-articular gel composition that promotes tissue and cartilage regeneration and a gel formulation in which the gel composition is provided in a carrier fluid. Said composition and formulation comprises sodium hyaluronate as well as the amino acids glycine and proline.

Description

[0001] INTRA- ARTICULAR GEL FOR TISSUE AND CARTILAGE REGENERATION

[0002] Technical Field

[0003] The present invention relates to an intra-articular gel composition which provides tissue and cartilage regeneration and to a gel formulation wherein the gel composition is presented in a carrier fluid. More particularly, the present invention relates to a composition and a formulation of an intra-articular gel with greatly improved pH stability.

[0004] Background of the Invention

[0005] Osteoarthritis (OA) is a degenerative joint disease thataffects the bone and soft tissues of the joint. Osteoarthritis, which belongs to the group of rheumatic diseases, is characterized as a type of arthritis (joint inflammation / arthritis) within this category. Osteoarthritis (OA) is a chronic and progressive disease that causes significant pain and difficulty in movement in patients.

[0006] Cartilage is a hard and slippery tissue that allows almost frictionless joint movement with the help of synovial fluid. Osteoarthritis is caused by the wear of the cartilage that acts as a shock absorber at the ends of the bones in the joints. In other words, in patients with osteoarthritis, wear and tear of the cartilage occurs and the cartilage tissue deteriorates over time. This leads to changes in the bone structure and degeneration of the connective tissues that hold the joint together and connect the muscles to the bone. In addition, inflammation of the joint capsule occurs.

[0007] Synovial fluid is a lubricating substance secreted by the synovial membrane that surrounds the area where two bones meetin the joints. This lubricating substance allows the jointto move easily. Under normal physiological conditions, the pH of the synovial fluid is alkaline and ranges from about 7.3 to 7.6. However, in patients with osteoarthritis, due to the accumulation of inflammatory metabolic products in and around the joint tissue, the pH of the synovial fluid moves towards the acidic range and can sometimes drop as low as 6.0 (Jin, T., et al. (2020). Intra-articular delivery of celastrol by hollow mesoporous silica nanoparticles for pH-sensitive anti-inflammatory therapy against knee osteoarthritis. J Nanobiotechnol 18, 94).

[0008] In the treatment of osteoarthritis, patients may experience joint tissue irritation and / or pain in the case the pH of therapeutic agents / compositions for the tissue and cartilage regeneration which are suitable for injection into the joint cavity differs significantly from the pH of the synovial fluid. In addition, the pH of therapeutic compositions for intra-articular administration is compatible with the low pH values in osteoarthritis patients, making it possible to maintain the efficacy and stability of the active ingredient in the composition also within the joint A review of intra-articular injections for the treatment of osteoarthritis suggests that various adverse effects such as pain, flare-ups, skin pigment changes, fat atrophy and joint infection may occur after intraarticular injection; and that compositions with a pH value of 7.4 or lower may stabilize the drug without activating inflammatory enzymes (lannitti, T., Lodi, D., & Palmieri, B. (2011). Intraarticular injections for the treatment of osteoarthritis: focus on the clinical use of hyaluronic acid. Drugs in R&D, 11(1), 13-27). In light of this information, it can be seen that it is important in the relevant technical field to keep the pH values of the formulations for intra-articular administration stable so that they are compatible with the pH values observed in the synovial fluids of osteoarthritis patients (e.g. around pH 7.0), which are low compared to the pH values observed in the synovial fluids of healthy people.

[0009] Examples of intra-articular fluid supplements commonly used in the early stage of cartilage degeneration include; mixtures comprising hyaluronic acid-sodium, hyaluronic acid- sodium / chondroitin sulfate or hyaluronic acid-sodium / chondroitin sulfate / N-acetylglucosamine.

[0010] Hyaluronate (also known as hyaluronic acid and hyaluronan) is a high molecular mass polysaccharide found naturally in the extracellular space of higher animals, especially in soft connective tissues. It is a linear polymer consisting of repeating monomers of n-acetyl glucosamine and glucuronic acid. Hyaluronate plays an important role in protecting the synovial joint by contributing to joint lubrication by buffering load transfer across articular surfaces (Schiavinato, A., Finesso, M., Cortivo, R., & Abatangelo, G. (2002). Comparison of the effects of intraarticular injections of Hyaluronan and its chemically cross-linked derivative (Hylan G-F20) in normal rabbit knee joints. Clinical and experimental rheumatology, 20(4), 445-454).

[0011] The most widely used form of hyaluronate, which is used for tissue regeneration in the treatment of osteoarthritis, is its sodium salt, sodium hyaluronate. Sodium hyaluronate is injected into the joint as part of osteoarthritis treatment to act as both a shock absorber and lubricant for the joint. By increasing the viscosity of the synovial fluid, it helps to lubricate, cushion and reduce pain in the joint Sodium hyaluronate has the following chemical structure:

[0012] Sodium hyaluronate

[0013] N- Acetyl glucosamine (GlcNAc), whose chemical structure is given below, is an amide derivative of the monosaccharide glucose. It is a secondary amide between glucosamine and acetic acid. Studies on mice by AR. Shikhman and colleagues have shown that N-acetyl-D-glucosamine plays an important role in the production of cartilage matrix components (Shikhman, A. R., et al. (2005). Chondroprotective activity of N-acetylglucosamine in rabbits with experimental osteoarthritis. Annals of the rheumatic diseases, 64(1 ), 89-94).

[0014] N- Acetylglucosamine

[0015] EP2886104A1 discloses an intra-articular gel formulation comprising sodium hyaluronate, N- acetyl glucosamine and chondroitin sulfate for the treatment of osteoarthritis.

[0016] Glycine has the chemical formula NH2-CH2-COOH and is the simplest stable amino acid. Glycine can be used in pharmaceutical formulations as a buffering agent, wetting agent, bulking agent, dietary supplement, tablet disintegrant and freeze-drying agent (Raymond C Rowe, Paul J, Sheskey & Marian E, Quinn (2009). Handbook of Pharmaceutical Excipients, 6th ed. RPS Publishing).

[0017] EP3544616A1 discloses that proline, another amino acid, is comprised in the formulation in order to stabilize the lyophilized formulation containing dalbavansin.

[0018] In addition to their use as excipients, the amino acids glycine and proline are important building blocks for collagen synthesis in joints. Osteoarthritis patients require large amounts of glycine, proline and lysine to restore normal levels of collagen synthesis, which is reduced due to osteoarthritis (de Paz-Lugo, P., et al. (2018), High glycine concentration increases collagen synthesis by articular chondrocytes in vitro: acute glycine deficiency could be an important cause of osteoarthritis. Amino acids, 50(10), 1357-1365).

[0019] EP1713464A2 relates to a method for increasing the flexibility of cartilage in an animal in need of such increase. The method involves administering to a mammal an effective amount of at least one of glycine and proline in order to increase cartilage flexibility.

[0020] EP2515897B1 relates to the use of an intra-articular gel containing a combination of glycine and proline in the treatment of osteoarthritis. The combination may also comprise a natural or synthetic viscosity-controlling polymer.

[0021] As can be seen, intra-articular gel formulations for the treatment of osteoarthritis are known in the relevant field. However, there is still a need for pH-stable intra-articular gel formulations for the treatment of tissue and cartilage regeneration in the relevant technical field. For this purpose, the present invention provides an intra-articular gel formulation having a stable pH that is compatible with the pH range of the synovial fluid observed in osteoarthritis patients. Said formulation increases patient compliance by preventing irritation and pain that may occur during and following administration. Furthermore, the present invention provides a composition and a formulation with high stability and thus extended shelflife. The compositions and formulations of the present invention are therefore advantageous in that they provide pH-stable products that maintain the efficacy and stability of the active ingredient and minimize the side effects.

[0022] Summary of the Invention

[0023] In one aspect, the invention provides an intra-articular gel composition, wherein said composition comprises the following components by weight:

[0024] - 0.1% to 2% NaH2PO4,

[0025] - 1% to 8% Na2HPO4,

[0026] - 2.5% to 15% NaCl,

[0027] - 20% to 40% N-acetyl-D- glucosamine,

[0028] - 7.5 % to 22.5 % glycine,

[0029] - 3.5% to 20% proline, and

[0030] - 15% to 45% sodium hyaluronate.

[0031] The intra-articular gel composition of the invention preferably comprises the following components by weight:

[0032] - 0.25% to 1.75% NaH2PO4, - 1.5% to 6% Na2HPO4,

[0033] - 3.5% to 12.5% NaCl,

[0034] - 22.5% to 37.5% N-acetyl-D- glucosamine,

[0035] - 10 % to 20 % glycine,

[0036] - 5% to 15% proline, and

[0037] - 20% to 40% sodium hyaluronate.

[0038] In a further preferred embodiment of the invention, the intra-articular gel composition of the invention comprises the following components by weight:

[0039] - 0.5% to 1.5% NaH2PO4,

[0040] - 2% to 5% Na2HPO4,

[0041] - 5% to 10% NaCl,

[0042] - 25% to 35% N-acetyl-D-glucosamine,

[0043] - 12.5 % to 17.5 % glycine,

[0044] - 7.5% to 12.5% proline, and

[0045] - 25 % to 35 % sodium hyaluronate.

[0046] The intra-articular gel composition of the invention may further be mixed with a carrier fluid, more preferably with water for injection, to form suitable formulations.

[0047] The intra-articular gel formulation of the invention preferably comprises between 5 mg and 40 mg of sodium hyaluronate in 1 ml of water. More preferably, the amount of sodium hyaluronate is 15 mg.

[0048] The amino acids proline and glycine comprised in the intra-articular gel composition or formulation of the invention are preferably in the L configuration.

[0049] In another aspect, the invention provides the intra-articular gel formulation of the invention for use in the prevention and / or treatment of tissue and / or cartilage degeneration in humans or animals. Said tissue and / or cartilage degeneration is preferably tissue and / or cartilage degeneration due to osteoarthritis.

[0050] Detailed Description of the Invention

[0051] The present invention relates to an intra-articular gel formulation with high pH stability. Said formulation comprises NaH2PO4(monosodium phosphate), Na2HPO4(disodium phosphate), NaCl (sodium chloride), N-acetyl-D- glucosamine, glycine, proline and sodium hyaluronate. The intra- articular gel formulation of the invention is effective in the prevention and / or treatment of cartilage degeneration, in particular in the treatment of osteoarthritis.

[0052] Accordingly, in one aspect, the present invention provides an intra-articular gel formulation, said formulation comprises the following components by weight:

[0053] - 0.1% to 2% NaH2PO4,

[0054] - 1% to 8% Na2HPO4,

[0055] - 2.5% to 15% NaCl,

[0056] - 20% to 40% N-acetyl-D- glucosamine,

[0057] - 7.5 % to 22.5 % glycine,

[0058] - 3.5% to 20% proline, and

[0059] - 15% to 45% sodium hyaluronate.

[0060] The formulation of the invention preferably comprises said components in the following percentages by weight:

[0061] - 0.25% to 1.75% NaH2PO4,

[0062] - 1.5% to 6% Na2HPO4,

[0063] - 3.5% to 12.5% NaCl,

[0064] - 22.5% to 37.5% N-acetyl-D- glucosamine,

[0065] - 10 % to 20 % glycine,

[0066] - 5% to 15% proline, and

[0067] - 20% to 40% sodium hyaluronate.

[0068] The formulation of the invention more preferably comprises said components in the following percentages by weight:

[0069] - 0.5% to 1.5% NaH2PO4,

[0070] - 2% to 5% Na2HPO4,

[0071] - 5% to 10% NaCl,

[0072] - 25% to 35% N-acetyl-D-glucosamine,

[0073] - 12.5% to 17.5% glycine,

[0074] - 7.5% to 12.5% proline, and

[0075] - 25% to 35% sodium hyaluronate.

[0076] The composition or formulation according to the present invention is intended to provide a product with high pH stability. Inventors of the present application have realized that combining the components of the composition in the percentages given above allows to obtain a product with high pH stability, as will be described in more detail below.

[0077] Accordingly, the composition of the present invention may comprise NaH2P04, Na2HP04, NaCl, N- acetyl-D-glucosamine, glycine, proline and sodium hyaluronate in any percentage within the above-mentioned weight percentage ranges, as to add up to 100%. For example, the intraarticular gel composition of the invention may comprise, by weight:

[0078] - 2% NaH2PO4,

[0079] - 4% Na2HPO4,

[0080] - 7% NaCl,

[0081] - 30% N-acetyl-D-glucosamine,

[0082] - 15% glycine,

[0083] - 10% proline, and

[0084] - 32% sodium hyaluronate.

[0085] The composition of the present invention may further be mixed with a carrier fluid, more preferably with water for injection, to obtain formulations of the invention. The amount of water for injection in which the components of the formulation are dissolved is preferably 1 ml.

[0086] The amino acids proline and glycine comprised in the intra-articular gel composition of the invention are preferably in the L configuration.

[0087] In another aspect, the invention provides the intra-articular gel composition or formulation according to the invention for use in the prevention and / or treatment of tissue and / or cartilage degeneration in humans or animals. Said tissue and / or cartilage degeneration is preferably tissue and / or cartilage degeneration due to osteoarthritis.

[0088] The molecular weight of the sodium hyaluronate comprised in the intra-articular gel composition of the invention is preferably in the range of 800 kDa to 3 MDa, more preferably in the range of 1 MDa to 2.5 MDa. The molecular weight of the sodium hyaluronate comprised in the formulation of the invention may be, for example, 800 kDa, 1 MDa, 1.5 MDa, 2 MDa, 2.5 MDa or 3 MDa.

[0089] The amount of sodium hyaluronate in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 5 mg to 40 mg, more preferably in the range of 7.5 mg to 30 mg. More specifically, the amount of sodium hyaluronate in 1 ml of water for injection is in the range of 10 mg to 20 mg. The amount of sodium hyaluronate in 1 ml of water for injection may be, for example, 10 mg, 13 mg, 15 mg, 18 mg or 20 mg. The amount of sodium hyaluronate in 1 ml of water for injection is most preferably 15 mg.

[0090] The amount of glycine in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 1 mg to 15 mg, more preferably in the range of 2.25 mg to 12.5 mg. More specifically, the amount of glycine in 1 ml of water for injection is in the range of 5 mg to 10 mg. The amount of glycine in 1 ml of water for injection may be, for example, 5 mg, 6 mg, 7.5 mg, 9 mg or 10 mg. The amount of glycine in 1 ml of water for injection is most preferably 7.5 mg. Said glycine is preferably L-glycine.

[0091] The amount of proline in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 1 mg to 12.5 mg, more preferably in the range of

[0092] 2.5 mg to 7.5 mg. More specifically, the amount of proline in 1 ml of water for injection is in the range of 3.75 mg to 6.5 mg. The amount of proline in 1 ml of water for injection may be, for example, 3.75 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg or 6.5 mg. The amount of proline in 1 ml of water for injection is most preferably 5 mg. Said proline is preferably L-proline.

[0093] The amount of N-acetyl-D-glucosamine in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 5 mg to 30 mg, more preferably in the range of 7.5 mg to 25 mg. More specifically, the amount of N-acetyl-D- glucosamine in 1 ml of water for injection is in the range of 10 mg to 20 mg. The amount of N- acetyl-D-glucosamine in 1 ml of water for injection may be, for example, 10 mg, 12.5 mg, 15 mg,

[0094] 17.5 mg. The amount of N-acetyl-D-glucosamine in 1 ml of water for injection is most preferably 15 mg. Said N-acetyl-D-glucosamine may be synthetic.

[0095] The amount of NalkPC (monosodium phosphate) in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 0.25 mg to 0.75 mg, more preferably in the range of 0.35 mg to 0.60 mg. The amount of NalkPC in 1 ml of water for injection is most preferably 0.45 mg.

[0096] The amount of Na2HPC>4 (disodium phosphate) in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 0.5 mg to 5 mg, more preferably in the range of 1 mg to 3.5 mg. The amount of Na2HPC>4 in 1 ml of water for injection is most preferably 2 mg.

[0097] The amount of NaCl (sodium chloride) in 1 ml of the intra-articular gel formulation of the invention (i.e. in 1 ml of water for injection) is preferably in the range of 2.5 mg to 7.5 mg, more preferably in the range of 3.5 mg to 6 mg. The amount of Naf^PC in 1 ml of water for injection is most preferably 4.5 mg.

[0098] The formulation according to the invention may comprise any combination of amounts of the components of the formulation corresponding to the ranges of amounts given above. The inventors have found that combining the components of the formulation in these ranges of amounts advantageously allows for both the high efficacy of the active ingredient and the high pH stability of the formulation.

[0099] Formulation

[0100] Example 1: Intra-Articular Gel Formulation in 1 ml of Water for Injection

[0101] The intra-articular gel formulation according to a preferred embodiment of the present invention is given in Table 1 below.

[0102] Table 1: Intra-Articular Gel Formulation in 1 ml of Water for Injection

[0103] The gel of Example 1 is an intra-articular matrix gel for the treatment of osteoarthritis, degenerative joint diseases and degenerative meniscal tears due to synovial fluid deficiency and for the promotion of cartilage regeneration. Said gel is administered into the joint by injection. pH Stability Studies

[0104] As explained in detail above, it is essential to keep intra-articular gel formulations stable at a preferred pH value. As a result of their observations, the inventors realized that the gel of the invention, which is provided for administering to osteoarthritis patients, should be maintained stable around pH 7.0. For the intra-articular gel formulations according to the invention comprising Hyaluronate at the concentrations indicated in Table 2 and Table 3 below (10 mg / ml, 13 mg / ml, 15 mg / ml, 18 mg / ml and 20 mg / ml), pH stability was evaluated under two different conditions: 1) pH Values for the Gel Samples without Glycine and Proline (Table 2) and 2) pH Values for the Gel Samples with Glycine and Proline (Table 3).

[0105] Table 2: pH Values of Glycine-Free and Proline-Free Gel Samples Before and After Sterilization

[0106] Table 3: pH Values of Glycine and Proline Containing Gel Samples Before and After Sterilization

[0107] Table 2 shows that the pH values of the gel formulations without glycine and proline were measured in the range of 6.92 to 7.28 before sterilization, wherein the formulations containing 10 mg / ml, 18 mg / ml and 20 mg / ml Hyaluronate were around 7.0. However, after autoclaving at 121°C for 15 minutes, a major deviation was observed in pH values for all concentrations and pH values even exceeded 7.4 for some concentrations.

[0108] Table 3 shows that the pH values of the gel formulations containing glycine and proline proved a high stability around pH 7.0 both before and after sterilization. In the light of this information, it can be seen that the gel formulation samples of the invention maintain their pH stability to a great extent before and after sterilization (autoclave) in case they contain glycine and proline. It is known in the relevant art that intra-articular compositions with a pH of 7.4 or higher may cause various adverse effects such as post-injection pain, flare-ups, skin pigment changes, fat atrophy and joint infection by activating the inflammatory enzymes, and adversely affect drug stabilization (lannitti, T., Lodi, D., & Palmieri, B. (2011). Intra-articular injections for the treatment of osteoarthritis: focus on the clinical use of hyaluronic acid. Drugs in R&D, 11(1), 13-27). Therefore, the formulations of the present invention, which have a highly stable pH value due to the glycine and proline components and their specific percentages in the formulation, increase patient compliance and allow the active ingredient to be injected with maximum efficiency due to their pH stability.

[0109] The intra-articular gel formulation of the present invention is stable due to its components and their specific weight percentages, the pH level of the formulation is substantially maintained around 7.0 and thus the formulation can be safely injected into the synovial fluid without causing irritation or pain to the patient.

Claims

CLAIMS1. An intra-articular gel composition, wherein the composition comprises the following components by weight:- 0.1% to 2% NaH2PO4,- 1% to 8% Na2HPO4,- 2.5% to 15% NaCl,- 20% to 40% N-acetyl-D- glucosamine,- 7.5 % to 22.5 % glycine,- 3.5% to 20% proline, and- 15% to 45% sodium hyaluronate.

2. The intra-articular gel composition according to claim 1, wherein the composition comprises the following components by weight:- 0.25% to 1.75% NaH2PO4,- 1.5% to 6% Na2HPO4,- 3.5% to 12.5% NaCl,- 22.5% to 37.5% N-acetyl-D- glucosamine,- 10 % to 20 % glycine,- 5% to 15% proline, and- 20% to 40% sodium hyaluronate.

3. The intra-articular gel composition according to claim 2, wherein the composition comprises the following components by weight:- 0.5% to 1.5% NaH2PO4,- 2% to 5% Na2HPO4,- 5% to 10% NaCl,- 25% to 35% N-acetyl-D-glucosamine,- 12.5 % to 17.5 % glycine,- 7.5% to 12.5% proline, and- 25% to 35% sodium hyaluronate.

4. The intra-articular gel composition according to any one of claims 1 to 3, wherein the amino acids glycine and proline comprised in the composition are in the L configuration.

5. An intra-articular gel formulation comprising the intra-articular gel composition according to any one of the preceding claims and water for injection.

6. The intra-articular gel formulation according to claim 5, wherein the formulation comprises between 5 mg and 40 mg of sodium hyaluronate in 1 ml of water for injection.

7. The intra-articular gel formulation according to claim 6, wherein the formulation comprises 15 mg sodium hyaluronate in 1 ml water for injection.

8. The intra-articular gel formulation according to claim 5, wherein the formulation comprises the following components by weight::Component Amount (mg)NaH2PO40.45Na2HPO42.00NaCl 4.50N-Acetyl-D-glucosamine 15.00L-glycine 7.50L-proline 5.00Sodium hyaluronate 15.

009. The intra-articular gel composition according to any one of claims 1 to 4 or the intra-articular gel formulation according to any one of claims 5 to 8 for use in the prevention and / or treatment of tissue and / or cartilage degeneration in humans or animals.

10. The composition or formulation for use according to claim 9, wherein said tissue and / or cartilage degeneration is tissue and / or cartilage degeneration due to osteoarthritis.

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